For an Escherichia coli cell to duplicate itself, three important processes must be completed. The cell must first accurately replicate its DNA. The newly formed chromosomes must then be resolved and segregated. An accurately placed septum at the midpoint of the cell must be made to ensure that each newly formed daughter cell receives a full complement of the cellular components. Over the past 30 years, studies investigating the E. coli cell cycle have identified many of the genes involved in these processes. However, identification of the internal signals that regulate these processes has been elusive.
At least 13 proteins are involved in the process of DNA replication (Kornberg and Baker, 1992.) The disclosures of each patent, patent application and publication cited or described in this document are hereby incorporated herein by reference, in their entirety. Initiation begins at a specific site, oriC, when DnaA binds to four dnaA boxes located within oriC (Kornberg and Baker, 1992). After loading of the other proteins (including DNA polymerase III, helicase, and primase) involved in the primosomal complex, replication proceeds bidirectionally until it reaches a site located approximately 180.degree. from oriC where replication terminates (ter). Termination requires two inverted ter sequences and the action of the Tus protein (Kornberg and Baker, 1992).
The era gene was discovered by sequencing downstream of the rnc gene in E. coli (Ahnn et al., 1986). The nomenclature for this gene and protein follows: Era represents the bacterial protein, era represents the bacterial gene, ERA represents the human protein, and ERA represents the human gene. The era gene is named for the initial identification of the gene from E. coli, (for E. coli ras). The era gene is a member of the rnc-era-recO rnc operon (Takiff et al., 1989). The rnc gene encodes the protein RNaseIII, which is an RNA endonuclease that plays a role in the processing of several RNA transcripts in E. coli (Bram et al., 1980; Gegenheimer and Apirion, 1981; King et al., 1986). Examples of RNA molecules processed by RNaseIII include rRNA transcripts, in which RNaseIII provides the initial cleavage in the maturation of precursor 30S rRNA to the functional 23S rRNA and 16S rRNA molecules, and the rnc operon, in which RNaseIII cleaves the transcribed message at a RNA hairpin found in the leader region of the operon (Bram et al., 1980; Gegenheimer and Apirion, 1981; King et al., 1986). This cleavage is important for the autoregulation of the rnc operon (Bardwell et al., 1989). The era gene in E. coli encodes a GTPase that has been proposed to play roles in the cell cycle, cell division, and growth rate (Ahnn et al., 1986; Gollop and March, 1991a; Gollop and March, 1991b). The RecO protein is involved in the RecF recombination pathway (Weinstock, 1987). Of the three genes, only the era gene is required for viability in E. coli (Takiff et al., 1989).
Presently the essential function of Era is unknown. Biochemical and cell biological studies of Era have been performed to attempt to elucidate the function of Era. The Era protein autophosphorylates in a GTP-dependent manner, however the function of the phosphorylated form of the protein in vivo is unknown (Sood et al., 1994). Immunoelectron microcopy to determine where Era localizes on the membrane showed that the protein is found near regions of potential division sites in E. coli (Gollop and March, 1991 a). The Era protein binds to the membrane in a GTP-dependent manner (Lin et al., 1994). The component of the membrane Era binds has not been discovered.
Morphological analysis of a strain in which the expression of era is repressed at low temperatures showed that cells formed long filaments in the absence of the Era protein (Gollop and March, 1991b). Based on these results and the localization of Era to potential division sites Gollop and March proposed that era may be involved in cell division (Gollop and March, 1991b). However, a similar type of strain construction where era is no longer expressed at high temperatures did not cause cells to filament, thus it is unclear whether decreased era expression results in an inhibition of cell division (Lerner and Inouye, 1991).
Proteins that comprise the GTPase superfamily have been discovered in organisms ranging from Escherichia coli to humans (Bourne et al., 1991). These proteins are turned "on" by the binding of GTP and "off" by the hydrolysis of GTP to GDP, thereby acting as a molecular switch (Bourne et al., 1991). The binding of GTP causes a conformational change in the protein that allows interaction with a target molecule (Bourne et al., 1991). GTPases are involved in diverse cellular processes such as signal transduction, protein translocation, and cell cycle regulation (Bourne et al., 1991). Much attention has focused on the small molecular weight GTPase ras and members of the ras subfamily of GTPases due to the identification of ras mutations in human cancer (Marshall, 1985).
Little is known about the functions of bacterial GTPases with respect to their possible role in the regulation of the bacterial cell cycle. However, GTPases are now being shown to play important roles in the cell cycles of bacteria. Recently it has been determined that a key cell division protein in E. coli, FtsZ, is a GTPase and requires GTP to polymerize in vitro (De Boer et al., 1992; RayChaudhuri and Park, 1992; Bramhill and Thompson, 1994; Mukherjee and Lutkenhaus, 1994). Also, the Obg protein in Bacillus subtilus is a GTPase that may play an important role in sporulation and DNA replication (Kok et al., 1994; Vidwans et al., 1995).
Era has been shown to be able to bind guanine nucleotides and is able to hydrolyze GTP to GDP (Ahnn et al., 1986; Chen et al., 1990). The protein also autophosphorylates in a GTP dependent manner in vitro (Sood et al., 1994). The function of this phosphorylated form of Era is unknown but it has been suggested that it is the active form of the protein (Sood et al., 1994). Binding of Era to the membrane has been demonstrated biochemically (Lin et al., 1994). Lin et al. have proposed that the regulation of Era binding to the membrane is determined within its GTPase activity (Lin et al., 1994).
The present invention describes the characterization of the sdgE class of suppressors. Both suppressors of this class are mutations affecting era, one of which (sdgE1) is a single point mutation within the era coding sequence. This is the first point mutation isolated within the chromosomal copy of era that affects the function of the Era protein without affecting other genes in the rnc operon. This mutation affects the GTPase activity of Era which reveals a cell cycle defect, at or near cell division.
The mechanism of cell division has been elusive in view of the lack of understanding of the roles of various genes thought to be involved in the process. In addition, the number of potential candidate drugs and agents for treating infectious diseases is vast. Accordingly, the development of a rational drug design, in addition to conventional antibiotic treatment, for treating infectious diseases has not progressed at an acceptable rate. With the continuing emergence of antibiotic-resistant pathogenic bacteria, efforts are needed to identify target genes and alternative methods of treating bacterial infectious diseases. To this end, the present invention satisfies this need by providing a method of screening for agents that delay a cell cycle. Accordingly, the present invention provides for methods of reducing or stopping the growth of infectious organisms and thus decreasing or eliminating infection. In addition, in view of the relation between bacterial era and human era, and by analogy to Ras, which Ras is mutated in several types of human cancers, the present invention also provides for a method of screening for anti-cancer agents.